Aligned amino-acid sequences of the putative hinge regions and the consensus motifs for PCNA binding derived from eukaryotic FEN1s. Residues within putative hinge regions are boxed and highlighted in red. Residues within the consensus motif for PCNA binding are in bold and highlighted in yellow.

A stereo view of the human FEN1–PCNA complex. Three FEN1 molecules are colored in blue (X), red (Y) and green (Z), and the three subunits of the PCNA trimer in yellow (A), cyan (B) and orange (C). The C-termini of FEN1 and PCNA are labeled. Metal ions bound to the active sites of FEN1 (X and Y) are shown in magenta. Proposed catalytic faces of FEN1 are indicated by arrows.

The hinge flexibility of human FEN1. (A) A stereo diagram showing three FEN1 molecules superimposed on PCNA (a gray model) as in Figure 6A, with a linear dsDNA in a wire model. The looping-out clamp regions are marked as dashed circles. The conceivable DNA model is superimposed at the center of PCNA. (B) Schematic model showing the orientation of the FEN1 core domains in (A).

Aligned amino-acid sequences of human and two archaeal FEN1s. The alignment was adjusted according to the three-dimensional structures. Secondary structure elements of human FEN1 (as in Figure 2A) are shown at the top, and residues whose electron densities were not observed in any of the three molecules are shown as dashed lines. Identical residues between human and archaeal FEN1s are shown in blue letters. The active-site residues which bind metal ions (pink) and the hinge region (red) are highlighted. The clamp region is boxed with black lines, and helices observed in the structures of two archaeal FEN1s (Hosfield et al, 1998; Hwang et al, 1998) are highlighted in light-blue. The PCNA-binding region of human FEN1 is boxed with blue lines, and those of human p21 have been aligned at the bottom. The X subscript indicates the number of followed core-domain residues.

The hinge region of human FEN1. (A) Superposition of the C-terminal tails from three human FEN1 molecules. The C-terminal tail plus helix α13 from three FEN1 molecules (in the same colors as in Figure 1) and the p21 peptide (magenta) illustrated as backbone-worm models are superimposed on part of the PCNA ring illustrated as a surface model. The orientation viewed is the same as in Figure 1 (PCNA-subunit A at the top). (B) Human FEN1 endonuclease activity enhanced by human PCNA. The deletion mutants of FEN1 are ΔG for deletion of G334, ΔGS for deletion of G334 and S335, ΔGST for deletion of residues 334–336, and ΔQGST for deletion of residues 333–336. The bar graph documents the cleavage (%) quantified from the gel in C with 10 pmol PCNA from the results of three independent experiments. Control experiments showed no significant changes in the activities without PCNA. (C) Reactions (10 μl) containing the SF substrate DNA (0.5 pmol), FEN1 (0.3 pmol) and PCNA (0, 1, 2, 5, 10 pmol). The mobility of the top band is 33 nucleotides (for the downstream primer) and that of the cleaved product is 20 nucleotides (for the flap DNA from the downstream primer).

Structure of human FEN1. All diagrams of FEN1 molecules are viewed from the same viewpoint as in (A). (A) Ribbon model of the molecular structure of human FEN1 (molecule Z). Two metal ions (M1 and M2) in the active site of molecule X are superimposed and depicted in magenta. (B) A stereo diagram showing superimposition of three human FEN1 molecules (X, Y and Z) including metal ions. Each molecule is shown in the same color as in Figure 1. (C) A stereo diagram showing superimposition of human FEN1 (green), M. jannachii FEN1 (Hwang et al, 1998) (violet) and P. furiosus FEN1 (Hosfield et al, 1998) (gold). The insertion sites at helix α0 and loop α10–α11 in human FEN1 are indicated by blue arrows and the deletion site at loop β6–β7 is shown as a red arrow. (D) Electrostatic molecular surfaces of human and M. jannachii FEN1s. Each surface was colored in the range <−10 to >+10k_BT, where k_B is the Boltzman constant and T is the temperature. The positive potential is shown in blue and the negative potential in red. (E) A closed-up stereo view of the active sites of human (green, molecule Y) and P. furiosus (gold) FEN1s. The orientation viewed is approximately that of a 90° rotation from (A) along the horizontal axis. The omit F_o–F_c electron density maps for the two metals (M1 and M2) are shown in blue (contoured at 5σ). The metal ions and side chain of Asp34 in P. furiosus FEN1 have not been deposited in PDB.

Interactions between FEN1 and PCNA. (A) Schematic depiction of interactions between FEN1 (green) and PCNA (yellow). The FEN1 hinge region is colored in red. Residues shown in blue participate in protein–protein interactions in one or two FEN1 molecules out of three: Ser25 in Y and Z, Arg29 in Z and X, and Lys30, Lys80 and Trp298 in X (see text). A tentative water molecule observed between molecules Z and C is indicated by a blue circle. (B) Human FEN1 endonuclease activity enhanced by human wild type (WT) and deletion mutant, Δ8 (254–261), Δ7 (255–261) and Δ5 (257–261), PCNAs. The mobility of the top band is 33 nucleotides (for the downstream primer) and that of the cleaved product is 20 nucleotides (for the flap DNA from the downstream primer). The size markers show 33 nucleotides, as indicated by an arrowhead with S and 20 nucleotides with P. The SF substrate DNA (0.5 pmol) was added into a solution containing FEN1 (0.3 pmol) and PCNA (0, 1, 2, 5, 10 pmol). Control experiments showed little detectable nuclease activity in the absence of PCNA. (C) The bar graph documents the cleavage (%) quantified from the gel with 10 pmol wild-type or mutant PCNA. Each cleavage was calculated from the results of three independent experiments. (D) Pull-down assays of wild-type and mutant recombinant PCNAs by FEN1(CHis). The presence of both FEN1(CHis) and PCNA bands in a single lane indicates the formation of an in vitro complex of the two proteins. The bar graph documents the relative binding by quantifying the protein amounts and normalizing with the beads-bound FEN1 amount. Each binding was calculated from the results of three independent experiments.